In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of an illuminating light beam is moved in an object plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in, for example, via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image.
In order to couple the excitation light of at least one light source into the microscope and to block out, from the light coming via the detection beam path from the specimen, the excitation light or excitation wavelength scattered and reflected at the specimen, it is also possible to use, instead of the beam splitter, an optical arrangement embodied as an acoustooptical component, for example as known from German Unexamined Application DE 199 06 757 A1.
A three-dimensional image is usually achieved by acquiring image data in layers, the path of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To make possible acquisition of image data in layers, the sample stage or the objective is shifted after a layer is scanned, and the next layer to be scanned is thus brought into the focal plane of the objective.
In many applications samples are prepared with several markers, for example several different fluorescent dyes. These dyes can be excited sequentially, for example using illuminating light beams that comprise different excitation wavelengths. Simultaneous excitation using an illuminating light beam that contains light of several excitation wavelengths is also usual. An arrangement having a single laser emitting several laser lines is known, for example, from European Patent Application EP 0 495 930, “Confocal microscope system for multi-color fluorescence.”. In practical use at present, such lasers are usually embodied as mixed-gas lasers, in particular as ArKr lasers.
The light power level of the illuminating light is subject, to fluctuations over time as a result of various effects, with negative repercussions in the context of sample examination.
One known method of compensating for short-term fluctuations in, for example, the illuminating light power level is based on dividing out a reference beam from the illuminating beam using a beam splitter, and using the ratio of the measured power levels of the reference and detected beams for image generation and calculation so that instantaneous power level fluctuations are thus eliminated. This is disclosed in G. J. Brakenhoff, Journal of Microscopy, Vol. 117, Pt. 2, November 1979, pp. 233–242. This method has certain disadvantages. For example, calculating out the laser power level fluctuations retrospectively upon image calculation is complex, and is not always an entirely satisfactory correction method. When a ratio is calculated from the measured power levels of the reference and detected light beams, offset components are not canceled out. In addition, the calculated scan image will wash out at locations with very low detected light power levels, since the signal-to-noise ratio no longer allows correct and unequivocal allocation of a hue or brightness to the scanned image point.
German Unexamined Application DE 100 33 269.2 A1 discloses an apparatus for coupling light into a confocal scanning microscope whose purpose is to compensate for or eliminate these fluctuations in illuminating light power level. The apparatus for coupling in light comprises an optically active component that serves in particular to select the wavelength and adjust the power level of the incoupled light. The apparatus is characterized in that in order to influence the incoupled light, the component serves as the adjusting element of a control system. A disadvantage of this apparatus is that the beam splitter which separates the illumination beam path from the detection beam path necessarily has a polarization- and wavelength-dependent reflectivity. The control operation as a result is laborious and complex, and necessitates complicated calibration measurements.
In German Unexamined Application DE 197 02 753 A1, it is proposed continuously to monitor the power level of the laser radiation, in particular of each individual laser line, that is coupled into the scanning head, and to compensate for fluctuations directly at the laser or using a downstream intensity modulator (ASOM, AOTF, EOM, shutter). The beam splitter problem already explained is relevant with respect to this disclosure as well.